Glutathione-S-transferases (GST) are a family of enzymes which catalyze the conjugation of glutathione, homoglutathione (hGSH) and other glutathione-like analogs via a sulfhydryl group, to a large range of hydrophobic, electrophilic compounds. The conjugation can result in detoxification of these compounds. GST enzymes have been identified in a range of plants including maize (Wosnick et al., Gene (Amst) 76 (1) (1989) 153-160; Rossini et al., Plant Physiology (Rockville) 112 (4) (1996) 1595-1600; Holt et al., Planta (Heidelberg) 196 (2) (1995) 295-302), wheat (Edwards et al., Pestic. Biochem Physiol. (1996) 54(2), 96-104), sorghum (Hatzios et al., J. Environ. Sci. Health, Part B (1996), B31(3), 545-553), arabidopsis (Van Der Kop et al., Plant Molecular Biology 30 (4) (1996), sugarcane (Singhal et al., Phytochemistry (OXF) 30 (5) (1991) 1409-1414), soybean (Flury et al., Physiologia Plantarum 94 (1995) 594-604) and peas (Edwards R., Physiologia Plantarum 98 (3) (1996) 594-604). GST's can comprise a significant portion of total plant protein, for example attaining from 1 to 2% of the total soluble protein in etiolated maize seedlings (Timmermann, Physiol Plant. (1989) 77(3), 465-71).
Glutathione S-transferases (GSTs; EC 2.5.1.18) catalyze the nucleophilic attack of the thiol group of GSH to various electrophilic substrates. Their functions and regulation in plants has been recently reviewed (Marrs et al., Annu Rev Plant Physiol Plant Mol Biol 47:127-58 (1996); Droog, F. J Plant Growth Regul 16:95-107, (1997)). They are present at every stage of plant development from early embryogenesis to senescence and in every tissue class examined. The agents that have been shown to cause an increase in GST levels have the potential to cause oxidative destruction in plants, suggesting a role for GSTs in the protection from oxidative damage. In addition to their role in the protection from oxidative damage, GSTs have the ability to nonenzymatically bind certain small molecules, such as auxin (Zettl et al., PNAS 91:689-693, (1994)) and perhaps regulate their bioavailability. Furthermore the addition of GSH to a molecule serves as an "address" to send that molecule to the plant vacuole (Marrs et al., Nature 375:397-400, (1995)).
GSTs have also been implicated in the detoxification of certain herbicides. Maize GSTs have been well characterized in relation to herbicide metabolism. Three genes from maize have been cloned: GST 29 (Shah et al., Plant Mol Biol 6, 203-211(1986)), GST 27 (Jepson et al., Plant Mol Biol 26:1855-1866, (1994)), GST 26 (Moore et al., Nucleic Acids Res 14:7227-7235 (1986)). These gene products form four GST isoforms: GST I (a homodimer of GST 29), GST II (a heterodimer of GST 29 and GST 27), GST III (a homodimer of GST 26), and GST IV (a homodimer of GST 27). GST 27 is highly inducible by safener compounds (Jepson (1994) supra; Holt et al., Planta 196:295-302, (1995)) and overexpression of GST 27 in tobacco confers alachlor resistance to transgenic tobacco (Jepson, personal communication). Additionally, Bridges et al. (U.S. Pat. No. 5,589,614) disclose the sequence of a maize derived GST isoform II promoter useful for the expression of foreign genes in maize and wheat. In soybean, herbicide compounds conjugated to hGSH have been detected and correlated with herbicide selectivity (Frear et al., Physiol 20: 299-310 (1983); Brown et al., Pest Biochem Physiol 29:112-120, (1987)). This implies that hGSH conjugation is an important determinant in soybean herbicide selectivity although this hypothesis has not been characterized on a molecular level.
Some efforts have been made to alter plant phenotypes by the expression of either plant or mammalian foreign GST genes or their promoters in mature plant tissue. For example, Helmer et al. (U.S. Pat No. 5,073,677) teach the expression of a rat GST gene in tobacco under the control of a strong plant promoter. Similarly, Jepson et al. (WO 97/11189) disclose a chemically inducible maize GST promoter useful for the expression of foreign proteins in plants; Chilton et al. (EP 256223) discuss the construction of herbicide tolerant plants expressing a foreign plant GST gene; and Bieseler et al. (WO 96/23072) teach DNA encoding GSTIIIc, its recombinant production and transgenic plants containing the DNA having a herbicide-tolerant phenotype.
Manipulation of nucleic acid fragments encoding soybean GST to use in screening in assays, the creation of herbicide-tolerant transgenic plants, and altered production of GST enzymes depend on the heretofore unrealized isolation of nucleic acid fragments that encode all or a substantial portion of a soybean GST enzyme.